Methods of analysis of radioactive material separated into solid and liquid phases

ABSTRACT

In analyses, particularly competitive assays, in which a radioactive element is partitioned between the two phases, the level of activity of one phase is measured while the two phases are in contact in the same vessel. This is achieved by using a specially shaped vessel and/or by accurately collimating the counter and screening it from part of the contents of the vessel.

United States Patent Glover et al.

METHODS OF ANALYSIS OF RADIOACTIVE MATERIAL SEPARATED INTO SOLID AND LIQUID PI-IASES Inventors: John Stuart Glover; Bryan Peter Shepherd, both of Amersham, England The Radiochemical Centre Limited, England Filed: Dec. 8, 1972 App]. No.: 313,263

Assignee:

Foreign Application Priority Data Dec. 16, 1971 United Kingdom 58566/71 US. Cl 250/303; 250/304 Int. Cl. G2lh 1/00 Field of Search 250/303, 302, 304

References Cited UNITED STATES PATENTS 3/1960 Jones et al. 250/304 Primary Examiner-Harold A. Dixon Attorney, Agent, or Firm-Wenderoth, Lind & Ponack [57] ABSTRACT In analyses, particularly competitive assays, in which a radioactive element is partitioned between the two phases, the level of activity of one phase is measured while the two phases are in contact in the same vessel. This is achieved by using a specially shaped vessel and/or by accurately collimating the counter and screening it from part of the contents of the vessel.

3 Claims, 8 Drawing Figures JIIIII PATENTED HAY I 31975 SHEET 10F 2 METHODS OF ANALYSIS OF RADIOACTIVE MATERIAL SEPARATED INTO SOLID AND LIQUID PHASES This invention relates to analyses of the kind in which a radioactive element or compound is partitioned between two phases and the proportion of the activity in each phase determined. Typical of such analyses are competitive assays of the kind in which an unknown amount of the compound to be assayed and a standard amount of a radioactively-labelled version of the compound compete for reaction with a standard amount of another reagent. This technique is widely used in the medical field for assaying hormones and other substances, using as the other reagent the antibody in immune systems or some other specific reactor in nonimmune or non-hormone systems.

The principle of the technique may be represented by the following scheme:

where C is the compound to be assayed,

C* is the labelled version of the compound R is the other reagent.

The amount of R is arranged to be insufficient to react with all of C C*. As the reaction is. at least to some extent reversible, an equilibrium is set up in which the ratio of [C*]/[C*]+[C*R] is determined by the amount of the unlabelled compound C which is present. If C* is separated from C*R and the level of activity of each separated part measured, then the value of the ratio is easily calculated. The amount of the unlabelled compound C can then be determined, in relative or absolute terms, by the use of standard preparation of compound C, to generate a calibration curve.

The technique is described, with examples, in a Review Paper by R. S. Yalow and S. A. Berson in IA- EASM124/l06, pages 455-481.

Separation of C* from C*R is generally effected in two stages. In the first stage, either C* or C*-R is caused to change phase for example by being precipitated from solution, or adsorbed on to an inert carrier, or passed into a water-immiscible phase. In the second stage, the two phase are removed from one another, e.g. by filtration, or, more usually, by centrifuging followed by decanting the supernatant liquor. With the two phases separated into different vessels, it is a simple matter to determine the level of activity in each.

The separation of the two phases by removal of one from the reaction tube into another vessel is timeconsuming, and gives rise to inaccuracies. In particular, the transfer of material from one vessel to another is rarely complete. It is an object of this invention to avoid the necessity for transfers of this kind.

The present invention provides a method of performing an analysis in which a radioactive element or compound is partitioned between two phases, which method comprises measuring the level of radioactivity of at least one of the two phases while that phase is in contact with the other phase.

At least a part of radioactive material in one phase has to be separated from the radioactive material in the other phase in order to determine the level of activity in the one phase. It is a feature of the invention that the two phases themselves are not removed from one an other, as by removal of one phase to a different vessel,

but remain in contact during measurement of the level of activity in one, or in both, of the phases.

The difficulty about measuring the level of activity in one phase, while the other phase remains in contact with it, lies in the difficulty of screening the device counting activity in the one phase from radiation emanating from the other phase. This invention envisages two ways of overcoming this difficulty, which may be employed either separately or, more usually, in combination:

I. The reaction tube may be specially shaped, for example, where the radioactive element is partitioned between a liquid and a heavier solid phase, as a centrifuge tube with a thin capillary at the bottom for the solid phase, so that the solid phase is separated by an appreciable distance from the bulk of the liquid phase in the body of the tube. (In the case where the radioactive element is partitioned between two liquid phases, a corresponding design of tube may be possible).

2. The counter may be accurately collimated and carefully screened from the phase whose activity is not being measured.

The method of this invention is particularly applicable in those cases where the radioactive tracer element involved is a soft gamma ray emitter, so that quite a thin layer of lead can be used for the screening. The commonly used tracer I is particularly suitable, as its soft gamma radiation is attenuated by 3 mm of lead to an insignificantly low level.

When there is used as the reaction vessel a centrifuge tube with a capillary at the bottom, the level of activity of the solid phase can be measured by screening off the body of the reaction vessel from the counter with only a very small error resulting from contamination by a tiny amount of liquid phase in the capillary; and the level of activity of the liquid phase in the body of the reaction vessel can be measured by screening off the capillary from the counter with only a very small error resulting from the absence of a tiny amount of the liquid phase in the capillary.

In many applications, an accurate measurement of the radioactivity in one, or in both, of the phases is not necessary, provided that the percentage error is not unduly large, and provided that it remains constant. Such a constant percentage error could, for example, give acceptable results in the normal procedure for radioimmunoassay, where a calibration curve is generated by reference to a standard preparation.

Features of the invention will now be further described with reference to the accompanying drawings, in which:

FIG. 1 is a perspective view of a conventional centrifuge tube and containing a solid in suspension in a liquid.

FIG. 2 is a perspective view of the centrifuge tube after centrifuging.

FIG. 3 is a perspective view of a partly shielded holder, with the cavity for a centrifuge tube shown dotted;

FIG. 4 is a perspective view of the holder with a centrifuge tube in position in it;

FIG. 5 is a perspective view of a shielded counting device, partly cut away to show a tube, containing solution and solid sediment, in position therein.

FIG. 6 is a sectional side elevation of the centrifuge tube and holder of FIG. 4 in position in the well of a sodium iodide scintillation crystal.

FIG. 7 is a sectional side elevation of the shielded tube of FIG. in position in the well of a sodium iodide scintillation crystal.

FIG. 8 is a sectional side elevation of a modification of the apparatus of FIG. 6 designed to measure the radioactivity of the solid phase but not the liquid phase.

In FIGS. 1 and 2, a centrifuge tube 10 is made of plastics material, has a cap 11, and contains a liquid phase 12 and a solid phase 13, either or both of which may be radioactive. In FIG. 2, the solid phase 13 has been concentrated in the conical tip 14 of the tube 10 by centrifuging.

In FIGS. 3 and 4, a partly shield holder 15, comprises a lead base 16 bonded to an annular upper portion 17 of plastics material. A centifuge tube fits in the holder so that the conical tip 14, and the solid material concentracted therein, is surrounded by the lead part of the holder.

The complete unit (holder plus centifuge tube 10) can be accommodated in the well of a sodium iodide scintillation crystal which forms part of an instrument for the measurement of radioactivity (a well-crystal counter). Screened from the counter by the lead base 16 of the holder 15 will be the solid phase 13 and the small proportion of the liquid phase 12 which is in the tip 14 of the centrifuge tube 10. The error in measurement, resulting from the fact that not quite all the liquid phase is visible in the counter, can generally be neglected unless very accurate measurements are required, and is in any case unimportant where levels of radioactivity of liquids in different tubes of the same design are being compared.

The system described with reference to FIGS. 1 to 4 is designed to measure the radioactivity of essentially the whole liquid phase in the centrifuge tube. However, when unknown samples are being measured against calibrating standards, measurement of the whole phase is not necessary. It is sufficient to ensure that the liquid volume is the same in different tubes being used in the experiment, and to measure the radioactivity of a constant volume, of the liquid phase. This constant volume need not be known, but it must form a constant proportion of the total liquid phase, as between different tubes used in the same experiment.

This principle is illustrated with reference to FIG. 5, where a counting device comprises a sodium iodide scintillation crystal (not shown) into the well of which lead shielding 18, 19 has been built. A tube 20, having a cap 21, and containing a liquid phase 22 up to a surface 23 and a solid sediment 24, fits snugly into the lead shielding. The upper and lower parts, 18 and 19 respectively, of the lead shielding limit the field of view of the counting device to a fixed column 25 of the liquid phase 22. Provided that the diameter of that column 25 is fixed (i.e. provided that the internal diametres of the tubes used in the experiment are all the same), and provided that the surface 23 of the liquid 22 is at the same level within the upper part 19 of the lead shielding in each tube the counter measures the radioactivity of a constant proportion of the total liquid phase, as between different tubes used in the same experiment. Alternatively, it is possible to operate the method with the liquid surface 23 within, rather than above the top of, the fixed column 25 in the field of view of the counting device. This alternative is not preferred, since it requires the surface 23 to be at accurately the same level as between different tubes in the same experiment.

Referring to FIG. 6, the centrifuge tube 10 in its holder 15 is placed in the well of a sodium iodide scintillation crystal 26. A scintillation counter 27 is positioned below the scintillation crystal. Radiation emitted by radioactive material in the liquid 12 passes through the plastics material 17 and gives rise to scintillations in the sodium iodide crystal 26. The scintillations are detected by the counter 27. Radiation from the solid deposit 13 is largely prevented from reaching the scintillation crystal 26 by the lead shielding 16.

Referring to FIG. 7, a well-type sodium iodide scintillation crystal 29 has lead shielding l8, 19 built into it in the manner described with reference to FIG. 5. Radiation emitted by radioactive material in the liquid 22 passes through the gap 25 between lead Shielding l8 and 19 and gives rise to scintillations in the sodium iodide crystal 29. The scintillations are detected by a counter 27, positioned below the scintillation crystal. Radiation from the remainder of the liquid 22 and from the solid 24 is prevented from reaching the scintillation crystal 29 by the lead shields 18, 19.

Referring to FIG. 8, the centrifuge tube 10 of FIG. 2 is placed in a holder resembling that of FIG. 3, except that in this case the base 30 is of plastics material and the annular upper part 31 is of lead. The tube and holder together are positioned in the well of a sodium iodide scintillation crystal 26. Surrounding the lower part of the scintillation crystal, on a level with the plastics base 30 of the holder, is a scintillation counter 32. Radiation emitted by radioactive material in the solid deposit 13 passes through the plastics material 30 and gives rise to scintillations in the sodium iodide crystal 26. These scintillations are detected by the counter 32. Radiation from the liquid 12 is largely prevented from reaching those parts of the scintillation crystal 26 which are observed by the counter 32 by means of the lead shielding 31.

Errors will be introduced if the tubes are not all of precisely the same internal diametre, or if the total volume of liquid is not precisely the same in every tube, but these errors are unlikely to be significant except for extremely accurate work.

EXAMPLE General Method.

An Insulin Immonoassay Kit manufactured by The Radiochemical Centre, based on the method of Hales and Randle (Lancet, i,200,l963; Biochem, J., 88, 137, 1963) was used to illustrate the use of the measuring system. An accredited serum was included in the assay as an unknown. This serum has been assayed in triplicate on more than 15 occasions using the conventional filtration method. The values obtained ranged from 43.0 to 58.5 milli units per ml with an overall mean of 49.7.

Procedure This was carried out exactly as laid down in the instruction sheet supplied with the kit, apart from the volumes taken to prepare the incubates: these being 300 ,u.l in place of the #1 suggested. The resulting larger volume enabled the existing shielded containers, which were designed for a nominal 1 ml volume, to be used without modification.

Insulin standards were prepared at 250, I00, 50, 25, I25, and zero uunits/ml. Triplicates at each concen tration were prepared by adding 300 pl aliquots to Eppendorf centrifuge tubes. Quadruplicates of the unknown sera were prepared by adding 300 ,ul of the sera to similar tubes.

To each of these tubes 300 ,ul of the binding reagent was added and mixed. The tubes were capped and kept at 4C for 6 hrs, whereupon they each received a similar 300 p.1 aliquot of the l25-l-lnsulin. The tubes were mixed, capped and kept at 4C for a further period of 18 hrs.

At the completion of this incubation the tubes were spun for 6 minutes in a Unipan 320" centrifuge at 14,500 r/m (14.000 g). They were then transferred to the shielded holders described above with reference to FIGS. 1 to 4 of the drawings, and each counted for 60 sees. in a Nuclear Enterprises auto-gamma counter type 831 1.

Results: Counts found in shielded containers.

in unknown scra determined for unknown' 68380 52.5 67943 50.5 52.2 uu/ml 68974 54.0 68185 52.0

Conclusion The result obtained for the insulin concentration of an accredited serum using the shielded measuring system compares very favourably with that obtained by the conventional membrane filtration.

As will be understood from the above description, the nature of the analysis is not material to this invention; immunoassays and similar competitive assays, as described above. are examples of suitable forms of analysis. The radioactive element involved is preferably a soft gamma ray emitter, but the nature of the two phases, and the method by which the radioactive element is partitioned between them, are not material. Likewise, any suitable means may be used for determining the level of activity in one or other (or both) of the phases.

We claim:

1. A method of performing an analysis in which a radioactive element or compound is partitioned between a liquid phase and a denser solid phase, which method comprises measuring the level of radioactivity of only one of the two phases while that phase is in contact with the other phase, wherein the measuring device is collimated and screened so as to measure the radioactivity of substantially the whole of the liquid phase but not of the solid phase.

2. A method of performing an analysis in which a radioactive element or compound is partitioned between a liquid phase and a denser solid phase, which method comprises measuring the level of radioactivity of only one of the two phases while that phase is in contact with the other phase, wherein the measuring device is collimated and screened so as to measure the radioactivity of a selected part only of the liquid phase.

3. A method of performing an analysis in which a radioactive element or compound is partitioned between a liquid phase and a denser solid phase, which method comprises measuring the level of radioactivity of only one of the two phases while that phase is in contact with the other phase, wherein the measuring device is collimated and screened so as to measure the radioactivity of the solid phase but not of the liquid phase.

* l l l l= 

1. A METHOD OF PERFORMING AN ANALYSIS IN WHICH A RADIOACTIVE ELEMENT OR COMPOUND IS PARTIONED BETWEEN A LIQUID PHASE AND A DENSER SOLID PHASE, WHICH METHOD COMPRISES MEASURING THE LECVEL OF RADIOACTIVITY OF ONLY ONE OF THE TWO PHASES WHILE THAT PHASE IS IN CONTACT WITH THE OTHER PHASE, WHEREIN THE MEASURING DEVICE IS COLLIMATED AND SCREENED SO AS TO MEASURE THE RADIOACTIVITY OF SUBSTANTIALLY THE WHOLE OF THE LIQUID PHASE BUT NOT OF THE SOLID PHASE.
 2. A method of performing an analysis in which a radioactive element or compound is partitioned between a liquid phase and a denser solid phase, which method comprises measuring the level of radioactivity of only one of the two phases while that phase is in contact with the other phase, wherein the measuring device is collimated and screened so as to measure the radioactivity of a selected part only of the liquid phase.
 3. A method of performing an analysis in which a radioactive element or compound is partitioned between a liquid phase and a denser solid phase, which method comprises measuring the level of radioactivity of only one of the two phases while that phase is in contact with the other phase, wherein the measuring device is collimated and screened so as to measure the radioactivity of the solid phase but not of the liquid phase. 